FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078089231> ?p ?o ?g. }

• W2078089231 endingPage "26249" @default.
• W2078089231 startingPage "26238" @default.
• W2078089231 abstract "Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G0/G1 cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G1 to S cell cycle progression. Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G0/G1 cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G1 to S cell cycle progression. Multiple factors contribute to the pathogenesis of atherosclerosis, a major health problem worldwide (1Ross R. N. Engl. J. Med. 1986; 314: 488-500Crossref PubMed Scopus (4022) Google Scholar). Proliferation of vascular smooth muscle cells (VSMCs), 2The abbreviations used are: VSMCvascular smooth muscle cellPDEphosphodiesterase3A-KOPDE3A-deficient3B-KOPDE3B-deficientMKPmitogen-activated protein kinase phosphataseCREBcAMP response element-binding proteinRbretinoblastomamCREBinactive CREBVP16 CREBactive CREBPKAprotein kinase A(Rp)-8-bromo-cAMPS(Rp)-8-bromoadenosine 3′,5′-cyclic monophosphorothioatePIphosphatidylinositol(Sp)-cAMPS(Sp)-adenosine 3′,5′-cyclic monophosphorothioatepBadphosphorylated BadATMataxia telangiectasia, mutatedATRATM- and Rad3-relatedSERCAsarco(endo)plasmic reticulum Ca2+-ATPase. primarily responsible for the maintenance of vascular tone (2Doran A.C. Meller N. McNamara C.A. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 812-819Crossref PubMed Scopus (629) Google Scholar), is a key event in the development of atherosclerotic lesions and postangioplasty restenosis (3Lafont A. Guzman L.A. Whitlow P.L. Goormastic M. Cornhill J.F. Chisolm G.M. Circ. Res. 1995; 76: 996-1002Crossref PubMed Scopus (291) Google Scholar). In a normal artery, VSMCs are in a non-proliferative, quiescent state and exhibit a well differentiated, “contractile” phenotype. After vascular injury, this differentiated phenotype is lost with a shift to a “synthetic” phenotype that is accompanied by entry into the cell cycle and proliferation (4Cai X. Expert Rev. Cardiovasc. Ther. 2006; 4: 789-800Crossref PubMed Scopus (42) Google Scholar). Evidence that pathological events in the vessel wall play an important role in atherosclerosis is increasing. Development of atherosclerosis may involve perturbation of the homeostatic balance between antiatherosclerotic signaling (nitric oxide (NO), atrial natriuretic peptide, and cyclic nucleotides) and proatherosclerotic signaling (tumor necrosis factor α (TNF-α) and angiotensin II) (5Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (6973) Google Scholar). Both endothelial cells and VSMCs are critical targets for inflammatory molecules, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, that are increased during progression of atherosclerosis (6Hansson G.K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1876-1890Crossref PubMed Scopus (729) Google Scholar), and are capable also of producing these molecules. In normal tissues with functional endothelium, NO and C-type natriuretic peptide produced by endothelial cells increase cyclic guanosine monophosphate (cGMP), which together with cyclic adenosine monophosphate (cAMP) and prostaglandins, limits inflammation and proliferation, and decrease vascular remodeling and atherosclerosis (7Koyama H. Bornfeldt K.E. Fukumoto S. Nishizawa Y. J. Cell. Physiol. 2001; 186: 1-10Crossref PubMed Scopus (82) Google Scholar). vascular smooth muscle cell phosphodiesterase PDE3A-deficient PDE3B-deficient mitogen-activated protein kinase phosphatase cAMP response element-binding protein retinoblastoma inactive CREB active CREB protein kinase A (Rp)-8-bromoadenosine 3′,5′-cyclic monophosphorothioate phosphatidylinositol (Sp)-adenosine 3′,5′-cyclic monophosphorothioate phosphorylated Bad ataxia telangiectasia, mutated ATM- and Rad3-related sarco(endo)plasmic reticulum Ca2+-ATPase. Phosphodiesterases (PDEs), which catalyze hydrolysis of cAMP/cGMP, belong to a complex and diverse superfamily of at least 11 structurally related, highly regulated, and functionally distinct gene families (PDE1–PDE11). These enzymes differ in their primary structures, affinities for cAMP and cGMP, responses to specific effectors, sensitivities to specific inhibitors, and mechanisms by which they are regulated (8Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (955) Google Scholar). Most PDE families comprise more than one gene, which yield multiple protein products via alternative mRNA splicing or utilization of different promoters and/or transcription initiation sites. The two PDE3 subfamilies, PDE3A and PDE3B, are encoded by closely related genes (9Maurice D.H. Palmer D. Tilley D.G. Dunkerley H.A. Netherton S.J. Raymond D.R. Elbatarny H.S. Jimmo S.L. Mol. Pharmacol. 2003; 64: 533-546Crossref PubMed Scopus (275) Google Scholar). PDE3A and PDE3B isoforms are expressed in VSMCs, but their exact role(s) is unclear in large part due to the lack of availability of specific inhibitors of individual PDE3 isoforms. The goals of this study were to understand the role(s) of PDE3A and PDE3B isoforms in VSMC function and to define mechanisms by which PDE3 isoforms might affect signaling pathways that regulate VSMC growth. Yan et al. (10Yan C. Miller C.L. Abe J.I. Circ. Res. 2007; 100: 489-501Crossref PubMed Scopus (80) Google Scholar) have shown that, in cultured cardiomyocytes, down-regulation of PDE3A with concomitant up-regulation of inducible cAMP early repressor was associated with increased cardiomyocyte apoptosis. Studies by Masciarelli et al. (11Masciarelli S. Horner K. Liu C. Park S.H. Hinckley M. Hockman S. Nedachi T. Jin C. Conti M. Manganiello V. J. Clin. Investig. 2004; 114: 196-205Crossref PubMed Scopus (202) Google Scholar) demonstrated that, in female mice, deletion of PDE3A resulted in infertility mainly due to arrest of oocyte meiosis via elevated cAMP/protein kinase A (PKA) signaling, which inhibited maturation-promoting factor. Several earlier studies demonstrated that cilostamide (a PDE3 inhibitor) and rolipram (a PDE4 inhibitor) increased accumulation of cAMP and inhibited arterial, lung, and mesangial smooth muscle cell growth and migration (12Netherton S.J. Maurice D.H. Mol. Pharmacol. 2005; 67: 263-272Crossref PubMed Scopus (109) Google Scholar, 13Inoue Y. Toga K. Sudo T. Tachibana K. Tochizawa S. Kimura Y. Yoshida Y. Hidaka H. Br. J. Pharmacol. 2000; 130: 231-241Crossref PubMed Scopus (35) Google Scholar, 14Chini C.C. Grande J.P. Chini E.N. Dousa T.P. J. Biol. Chem. 1997; 272: 9854-9859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Matousovic K. Grande J.P. Chini C.C. Chini E.N. Dousa T.P. J. Clin. Investig. 1995; 96: 401-410Crossref PubMed Scopus (77) Google Scholar). Thus, we reasoned that PDE3A-deficient (3A-KO) and PDE3B-deficient (3B-KO) mice might be valuable models in which to dissect and reconstitute signaling pathways downstream of cAMP that could elucidate mechanisms controlling cell division and mitogenesis in other types of cells, including VSMCs. Our results indicate that PDE3A depletion inhibited mitogen-induced VSMC proliferation via two complementary signaling pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1, which interfered with activation of MAPK signaling, and PKA/CREB-induced elevation of p21, leading to cell cycle arrest in G0/G1, as well as by increased accumulation of p53, which induces mitogen-activated protein kinase phosphatase 1 (MKP-1), p21, and WIP1, leading to inhibition of G1 to S cell cycle progression. All materials and reagents used were commercially available as detailed in the supplemental Materials. 3A-KO mice were generated by targeted disruption of exon 13, which encodes a portion of the second putative metal-binding site in the PDE3A catalytic domain (11Masciarelli S. Horner K. Liu C. Park S.H. Hinckley M. Hockman S. Nedachi T. Jin C. Conti M. Manganiello V. J. Clin. Investig. 2004; 114: 196-205Crossref PubMed Scopus (202) Google Scholar). Generation of 3B-KO mice (MPDE3BSvJ129) was also described earlier (16Choi Y.H. Park S. Hockman S. Zmuda-Trzebiatowska E. Svennelid F. Haluzik M. Gavrilova O. Ahmad F. Pepin L. Napolitano M. Taira M. Sundler F. Stenson Holst L. Degerman E. Manganiello V.C. J. Clin. Investig. 2006; 116: 3240-3251Crossref PubMed Scopus (139) Google Scholar). Mice were maintained and studies were performed in accordance with protocols approved by the Animal Care and Use Committee at NHLBI, National Institutes of Health. Male 3A-KO (PDE3A−/−C57BL/6J-129/SvJ) mice from the F1 generation, 3A-KO (PDE3A−/−C57BL/6J) backcrosses from the F8 generation, and 3B-KO mice (MPDE3BSvJ129) from the F8 generation at the age of 8–12 weeks were used for isolation of primary VSMCs. Similar results were obtained with F1 and F8 generations of 3A-KO mice with respect to VSMC growth inhibition and alterations in signaling proteins. However, VSMCs grown from male F1 generation 3A-KO mice were largely epithelioid in shape, whereas VSMCs grown from 3A-WT littermates were largely spindle-shaped (see supplemental Fig. 1). The epithelioid morphology of 3A-KO VSMCs grown from F1 generation 3A-KO mice reverted to a spindle shape upon eight backcrosses with C57BL/6J mice (supplemental Fig. 1). Thus, differences in morphology were not causally related to differences in growth, MAPK, and cell signaling pathways between 3A-WT and 3A-KO VSMCs grown from F1 generation mice. VSMCs isolated by collagenase digestion of the aortic media layer as described (17Begum N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar) were not contaminated with fibroblasts or endothelial cells as evidenced by >99% positive immunostaining with fluorescein isothiocyanate-conjugated antibodies against smooth muscle α-actin (Sigma). VSMCs maintained in α-minimum Eagle's medium containing 10% fetal bovine serum and 1% antibiotic/antimycotic were grown to confluence and studied at passage 5 for all experiments. Confluent, serum-starved VSMCs were treated with PDGF-BB (10 ng/ml) or insulin (100 nm) alone or with insulin followed by PDGF and extracted with lysis buffer. Proteins (50 μg) were separated by SDS-PAGE followed by Western immunoblot analysis and detected with enhanced chemiluminescence reagent and imaging with a charge-coupled device camera (LAS 1000 Plus, Fuji, Tokyo, Japan). ECL signals in the linear range were quantitated by densitometry as described in the figure legends. Cell proliferation was quantified by using BrdU cell proliferation assay kits (Roche Applied Science). DNA synthesis was assessed by [3H]thymidine incorporation as described earlier (18Jacob A. Smolenski A. Lohmann S.M. Begum N. Am. J. Physiol. Cell Physiol. 2004; 287: C1077-C1086Crossref PubMed Scopus (21) Google Scholar, 19Begum N. Song Y. Rienzie J. Ragolia L. Am. J. Physiol. Cell Physiol. 1998; 275: C42-C49Crossref PubMed Google Scholar). For cell cycle analysis, VSMCs were incubated with PDGF for 24 h, harvested with trypsin, and stained (1 × 106 cells) with 100 μg/ml propidium iodide for 12 h in the dark at 4 °C. Fractions of cells present in each phase of the cell cycle (G0/G1, S, and G2/M) were determined by flow cytometry using a BD FACStar flow cytometer and ModiFit software. Cell cycle-specific gene expression was analyzed by Oligo GEArray mouse cell cycle arrays (SABiosciences, Frederick, MD) representing 112 genes critical to cell cycle regulation. RNA (1 μg) isolated from quiescent 3A-WT and 3A-KO VSMCs was used to synthesize biotin-16-UTP-labeled target cRNA probes, which were hybridized to array membranes, according to the manufacturer's directions. Chemiluminescent signals were detected, and the ECL images were analyzed using GEArray Expression Analysis suite software. Data were normalized to values for housekeeping genes. PKA activity was assayed in VSMC extracts using Upstate Biotechnology Inc. (Upstate, NY) kits with Kemptide as substrate and [γ-32P]ATP without or with protein kinase inhibitor peptide. PKC and calcium/calmodulin-dependent protein kinase activities were inhibited by including inhibitor peptide mixture in the assay mixture. PDE3 and PDE4 activities in VSMC extracts (5-μl samples) were measured in the presence and absence of cilostamide (PDE3 inhibitor) and rolipram (PDE4 inhibitor) using the SPA PDE assay kit (Amersham Biosciences) in accord with the manufacturer's instructions. The concentration of cAMP was 1 μm with 0.05 μCi of [3H]cAMP/assay. Subconfluent VSMCs were transfected according to the manufacturer's protocol with 100 nm scrambled RNAi as a control or with 100 nm PDE3A siRNA (sc-41593, Santa Cruz Biotechnology, Inc.), which is a pool of three target-specific 20–25-nucleotide siRNAs, or with 100 nm siGenome ON-TARGETplus SMARTpool mouse TRP53 (L-040642–00, Dharmacon), which is a pool of four target-specific siRNAs. After 48 h, VSMCs were serum-starved, incubated with PDGF, and examined for reductions in PDE3A or p53 protein by Western blotting. [3H]Thymidine incorporation was used to quantify DNA synthesis as described (17Begum N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar). Protein concentrations in cell homogenates were quantitated by the bicinchoninic acid method (20Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar). Unless otherwise noted, all data are reported as means ± S.E. of values from at least four or five experiments performed in duplicate. Analysis of variance followed by Dunnett's test was used to compare mean values for different treatments or groups. A p value <0.05 was considered statistically significant. As seen in supplemental Fig. 2A, Western blot analyses with isoform-specific antibodies confirmed the absence of PDE3A and PDE3B isoforms in primary VSMCs grown from aortas of 3A-KO and 3B-KO mice, respectively, and their presence in WT counterparts. Of note, there was a compensatory increase in PDE3A protein in 3B-KO VSMCs but no such increase in PDE3B in 3A-KO VSMCs. As seen in supplemental Fig. 2B, analysis of PDE enzymatic activities indicated that most of the cAMP PDE hydrolytic activity in VSMCs was due to PDE3 and PDE4 isoforms based on inhibition of PDE activity by cilostamide and rolipram (21Phillips P.G. Long L. Wilkins M.R. Morrell N.W. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 288: L103-L115Crossref PubMed Scopus (81) Google Scholar). In 3A-KO VSMCs, PDE3A deletion resulted in an ∼70% decrease in PDE3 activity, whereas PDE4 remained unaffected. 3B-WT VSMCs exhibited ∼50 and ∼30% increases in total PDE and PDE3 activities, respectively, when compared with 3A-WT VSMCs. In 3B-KO VSMCs, PDE3B deletion resulted in an ∼43% decrease in PDE3 activity. The magnitude of decrease in PDE3 activity, ∼43% in 3B-KO VSMCs, was less than the corresponding decrease of ∼70% in 3A-KO VSMCs (supplemental Fig. 2B). This may be due to the observed increase in PDE3A protein in 3B-KO VSMCs. Cellular PKA activity in lysates of VSMCs from aortas of 3A-KO and 3B-KO mice was assayed as a measure of assessing altered cAMP signaling due to changed PDE3 expression. Basal PKA activity in 3A-KO VSMCs was more than twice that in 3A-WT cells (9.91 ± 1.5 versus 3.7 ± 0.59 pmol of phosphate incorporated into Kemptide/mg of protein/min; mean ± S.E., n = 4), whereas no such increase was observed in 3B-KO VSMCs (3.72 ± 0.68 versus 3.76 ± 0.47 pmol/mg of protein/min). This absence of increased PKA activity in 3B-KO VSMCs may also be due to, at least in part, the increased expression of PDE3A protein in 3B-KO VSMCs. Acute stimulation with PDGF for 5 min increased PKA activity in VSMCs by 60–70% over basal levels in all VSMCs (3A-WT, 6.3 ± 0.88; 3A-KO, 16.2 ± 1.83; 3B-WT, 6.5 ± 0.59; 3B-KO, 6.2 ± 0.59). PKA activity was completely inhibited when PKA peptide inhibitor was included in vitro during the enzyme assays and partially inhibited when VSMCs were incubated with (Rp)-8-bromo-cAMPS, a cAMP analog that inhibits PKA (data not shown). VSMC proliferation is known to play a crucial role in the development of restenosis after balloon angioplasty and in the progression of fatty streaks to atherosclerotic plaques (2Doran A.C. Meller N. McNamara C.A. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 812-819Crossref PubMed Scopus (629) Google Scholar). To determine the role of PDE3 isoforms in regulation of VSMC proliferation and growth, we measured [3H]thymidine incorporation into DNA after treatment of VSMCs with PDGF or serum. As seen in Fig. 1A, PDGF- or serum-induced DNA synthesis was increased 4–6-fold in 3A-WT, 3B-WT, and 3B-KO, but not in 3A-KO, VSMCs. Results were similar when growth rates were measured by colorimetric assays using anti-BrdU antibodies to mark proliferating cells (data not shown). Thus, in contrast to 3B-KO VSMCs, which were responsive to mitogenic stimuli, PDE3A depletion was accompanied by markedly less proliferation in response to mitogenic stimuli. To confirm a major role for PDE3A in regulation of VSMC proliferation, PDE3A was depleted in 3B-WT and 3B-KO VSMCs using murine PDE3A siRNA (sc-41593 (Santa Cruz Biotechnology, Inc.), which is a pool of three target-specific 20–25-nucleotide siRNAs). Transfection with PDE3A siRNA resulted in 65–70% depletion of cellular PDE3A (Fig. 1B) and significantly decreased basal and PDGF-induced DNA synthesis in 3B-WT and 3B-KO VSMCs when compared with VSMCs transfected with scrambled siRNA (Fig. 1C). Thus, PDE3A depletion in 3B-WT and 3B-KO VSMCs is accompanied by a marked reduction in VSMC proliferative capacity in response to mitogenic stimuli. The effect of PDE3A deletion on cell cycle progression was assessed by FACS analysis of propidium iodide-labeled VSMCs (Fig. 2). In the basal state, a large fraction of 3A-WT and 3A-KO VSMCs accumulated in G0/G1 phase with a small fraction of each in S phase (Fig. 2). Incubation of 3A-WT VSMCs with PDGF (24 h) induced cell cycle progression into S phase, significantly decreasing the percentage of cells in G0/G1 and concomitantly increasing the numbers of cells in S phase (2.5-fold) and in G2/M phase (6-fold) (Fig. 2, top right). In contrast, 3A-KO VSMCs exhibited markedly decreased percentages of cells in G1/S and G2/M in response to PDGF (Fig. 2, bottom right; 86.2 ± 3.1% in G0/G1, 9.4 ± 1.4% in S phase, and 4.4 ± 1.0% in G2/M phase). These effects strongly correlated with the inhibition of mitogen-induced DNA synthesis in 3A-KO VSMCs (Fig. 1). Overall, these findings suggested that PDE3A deletion increased PKA activity and inhibited VSMC proliferation by blocking mitogen-induced G1 → S phase and G2 → M phase progression. This observation prompted us to investigate potential alterations in upstream and downstream signaling pathways that regulate cell cycle proteins involved in VSMC growth and proliferation. In VSMCs, growth factors activate two major mitogenic signaling pathways in early G1, MAPK/ERK and PI 3-kinase signaling pathways (22Nelson P.R. Yamamura S. Mureebe L. Itoh H. Kent K.C. J. Vasc. Surg. 1998; 27 (1998): 117-125Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Isenović E.R. Kedees M.H. Tepavcević S. Milosavljević T. Korićanac G. Trpković A. Marche P. Cardiovasc. Hematol. Disord. Drug Targets. 2009; 9: 172-180Crossref PubMed Scopus (41) Google Scholar, 24Pagès G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar). MAPK/ERK activation is required for cell cycle initiation (G0-G1) as well as cell cycle progression (22Nelson P.R. Yamamura S. Mureebe L. Itoh H. Kent K.C. J. Vasc. Surg. 1998; 27 (1998): 117-125Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Isenović E.R. Kedees M.H. Tepavcević S. Milosavljević T. Korićanac G. Trpković A. Marche P. Cardiovasc. Hematol. Disord. Drug Targets. 2009; 9: 172-180Crossref PubMed Scopus (41) Google Scholar, 24Pagès G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar). Phosphorylation/activation of upstream kinases (MEK and Raf-1) results in activation of MAPK, leading to phosphorylation of downstream kinases and enhanced expression of several immediate early gene products, which promote cell cycle progression through the restriction point of the cell cycle (25Ducommun B. Semin. Cell Biol. 1991; 2: 233-241PubMed Google Scholar). To test whether growth inhibition in 3A-KO VSMCs might be related to alterations in MAPK/ERK signaling, immunoblot analyses were performed on cell extracts derived from VSMCs acutely stimulated for 5 min with PDGF, insulin, or a combination of both (Fig. 3, top). Insulin was included along with PDGF to determine whether it potentiated PDGF-induced MAPK/ERK phosphorylation/activation, which is required for proliferation of various cells, including VSMCs (22Nelson P.R. Yamamura S. Mureebe L. Itoh H. Kent K.C. J. Vasc. Surg. 1998; 27 (1998): 117-125Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Isenović E.R. Kedees M.H. Tepavcević S. Milosavljević T. Korićanac G. Trpković A. Marche P. Cardiovasc. Hematol. Disord. Drug Targets. 2009; 9: 172-180Crossref PubMed Scopus (41) Google Scholar, 24Pagès G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar, 25Ducommun B. Semin. Cell Biol. 1991; 2: 233-241PubMed Google Scholar). PDGF treatment rapidly increased phosphorylation of ERK1 and ERK2 by severalfold in 3A-WT, 3B-WT, and 3B-KO VSMCs. In contrast, PDGF-induced ERK1 and ERK2 phosphorylations were markedly reduced in 3A-KO VSMCs, reflecting deficient enzyme activation. Insulin treatment alone or in combination with PDGF did not increase ERK phosphorylation. Densitometric analyses revealed that these reductions in ERK phosphorylations were not due to reductions in ERK protein (Fig. 3, bottom). The time course of PDGF-induced ERK phosphorylation revealed persistent reductions in 3A-KO VSMCs at all times studied (data not shown). Incubation of 3B-KO and 3B-WT VSMCs with cAMP agonists ((Sp)-cAMPS; 100 μm) for 30 min also abrogated subsequent PDGF-induced ERK phosphorylation (supplemental Fig. 3). p38 MAPK phosphorylation was also reduced in 3A-KO VSMCs (data not shown). Earlier studies with cilostamide (a PDE3 inhibitor) and cAMP agonists have shown similar reductions in ERK phosphorylation (12Netherton S.J. Maurice D.H. Mol. Pharmacol. 2005; 67: 263-272Crossref PubMed Scopus (109) Google Scholar, 13Inoue Y. Toga K. Sudo T. Tachibana K. Tochizawa S. Kimura Y. Yoshida Y. Hidaka H. Br. J. Pharmacol. 2000; 130: 231-241Crossref PubMed Scopus (35) Google Scholar, 14Chini C.C. Grande J.P. Chini E.N. Dousa T.P. J. Biol. Chem. 1997; 272: 9854-9859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Matousovic K. Grande J.P. Chini C.C. Chini E.N. Dousa T.P. J. Clin. Investig. 1995; 96: 401-410Crossref PubMed Scopus (77) Google Scholar, 26Cheng J. Thompson M.A. Walker H.J. Gray C.E. Diaz Encarnacion M.M. Warner G.M. Grande J.P. Am. J. Physiol. Renal Physiol. 2004; 287: F940-F953Crossref PubMed Scopus (28) Google Scholar) that were accompanied by growth inhibition. Studies by Inoue et al. (13Inoue Y. Toga K. Sudo T. Tachibana K. Tochizawa S. Kimura Y. Yoshida Y. Hidaka H. Br. J. Pharmacol. 2000; 130: 231-241Crossref PubMed Scopus (35) Google Scholar) have shown that cilostamide, which increased VSMC cAMP content, inhibited DNA synthesis stimulated by fetal calf serum but did not affect VSMC migration stimulated by PDGF. This earlier report (13Inoue Y. Toga K. Sudo T. Tachibana K. Tochizawa S. Kimura Y. Yoshida Y. Hidaka H. Br. J. Pharmacol. 2000; 130: 231-241Crossref PubMed Scopus (35) Google Scholar) suggested that cilostamide suppressed rat arterial intimal hyperplasia in single and double balloon injury models presumably by inhibiting proliferation rather than migration of VSMCs. PI 3-kinase signaling pathways are also essential for cell proliferation, cell survival, and differentiation (27Duan C. Bauchat J.R. Hsieh T. Circ. Res. 2000; 86: 15-23Crossref PubMed Scopus (150) Google Scholar). As shown in Fig. 4 (top), insulin or PDGF rapidly increased phosphorylation of AktSer-473, a target of PI 3-kinase signaling. AktSer-473 phosphorylation was similar in 3A-KO, 3A-WT, 3B-WT, and 3B-KO VSMCs (Fig. 4, bottom). Thus, PI 3-kinase/Akt signaling appeared to be preserved in 3A-KO VSMCs. Additional studies indicated that pBad phosphorylation in 3A-KO VSMCs was comparable with that in WT cells (data not shown). Because Akt inhibits apoptosis by phosphorylation and inactivation of several proteins, including FoxO1, Bad, and caspase-9, preservation of PI3K/Akt signaling in 3A-KO VSMCs supported the hypothesis that the reduction in proliferation of 3A-KO VSMCs was not likely to be due to excessive apoptosis. cAMP/PKA signaling is known to increase phosphorylation of Raf-1 at Ser-259, which results in inhibition of Raf-1 kinase activity (28Dhillon A.S. Pollock C. Steen H. Shaw P.E. Mischak H. Kolch W. Mol. Cell. Biol. 2002; 22: 3237-3246Crossref PubMed Scopus (190) Google Scholar) and consequently inhibition of ERK phosphorylation/activation. As seen in Fig. 5 (top), basal Raf-1Ser-259 inhibitory site phosphorylation was elevated more than 3-fold in 3A-KO VSMCs compared with that in 3A-WT or 3B-KO VSMCs (Fig. 5, bottom, 3A-KO basal versus 3A-WT or 3B-KO basal) consistent with increased PKA activity in 3A-KO VSMCs. Treatment with insulin or PDGF for 5 min increased Raf-1 phosphorylation in all VSMCs. However, phosphorylation of Raf-1Ser-259 by insulin or PDGF was higher in 3A-KO VSMCs compared with that in 3A-WT or 3B-KO VSMCs. In all VSMCs, treatment with insulin for 5 min followed by incubation with PDGF for 5 min reverted the phosphorylation state to basal or near-basal levels. The observed transie" @default.
• W2078089231 created "2016-06-24" @default.
• W2078089231 creator A5017542878 @default.
• W2078089231 creator A5037955785 @default.
• W2078089231 creator A5081356989 @default.
• W2078089231 date "2011-07-01" @default.
• W2078089231 modified "2023-10-17" @default.
• W2078089231 title "Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins" @default.
• W2078089231 cites W1590358488 @default.
• W2078089231 cites W1967880683 @default.
• W2078089231 cites W1971863225 @default.
• W2078089231 cites W1972305847 @default.
• W2078089231 cites W1972431492 @default.
• W2078089231 cites W1982249395 @default.
• W2078089231 cites W1985935940 @default.
• W2078089231 cites W1989710827 @default.
• W2078089231 cites W1996571204 @default.
• W2078089231 cites W2023472206 @default.
• W2078089231 cites W2032575292 @default.
• W2078089231 cites W2033030646 @default.
• W2078089231 cites W2045876058 @default.
• W2078089231 cites W2050611911 @default.
• W2078089231 cites W2055964034 @default.
• W2078089231 cites W2061755142 @default.
• W2078089231 cites W2064129448 @default.
• W2078089231 cites W2066963386 @default.
• W2078089231 cites W2070038803 @default.
• W2078089231 cites W2079917183 @default.
• W2078089231 cites W2086109356 @default.
• W2078089231 cites W2090193505 @default.
• W2078089231 cites W2093095584 @default.
• W2078089231 cites W2094516179 @default.
• W2078089231 cites W2097638388 @default.
• W2078089231 cites W2104479866 @default.
• W2078089231 cites W2106480129 @default.
• W2078089231 cites W2106517675 @default.
• W2078089231 cites W2109538717 @default.
• W2078089231 cites W2120543791 @default.
• W2078089231 cites W2128251228 @default.
• W2078089231 cites W2130919931 @default.
• W2078089231 cites W2132347940 @default.
• W2078089231 cites W2133970318 @default.
• W2078089231 cites W2140488837 @default.
• W2078089231 cites W2145708730 @default.
• W2078089231 cites W2146917818 @default.
• W2078089231 cites W2147812298 @default.
• W2078089231 cites W2151425122 @default.
• W2078089231 cites W2152271058 @default.
• W2078089231 cites W2153139344 @default.
• W2078089231 cites W2158324497 @default.
• W2078089231 cites W2158438063 @default.
• W2078089231 cites W2165474821 @default.
• W2078089231 cites W2165569527 @default.
• W2078089231 cites W2166127640 @default.
• W2078089231 cites W2339699486 @default.
• W2078089231 cites W2342317553 @default.
• W2078089231 cites W3196550503 @default.
• W2078089231 cites W343184009 @default.
• W2078089231 cites W4232534952 @default.
• W2078089231 doi "https://doi.org/10.1074/jbc.m110.214155" @default.
• W2078089231 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3138244" @default.
• W2078089231 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21632535" @default.
• W2078089231 hasPublicationYear "2011" @default.
• W2078089231 type Work @default.
• W2078089231 sameAs 2078089231 @default.
• W2078089231 citedByCount "60" @default.
• W2078089231 countsByYear W20780892312012 @default.
• W2078089231 countsByYear W20780892312013 @default.
• W2078089231 countsByYear W20780892312014 @default.
• W2078089231 countsByYear W20780892312015 @default.
• W2078089231 countsByYear W20780892312016 @default.
• W2078089231 countsByYear W20780892312017 @default.
• W2078089231 countsByYear W20780892312018 @default.
• W2078089231 countsByYear W20780892312019 @default.
• W2078089231 countsByYear W20780892312020 @default.
• W2078089231 countsByYear W20780892312021 @default.
• W2078089231 countsByYear W20780892312022 @default.
• W2078089231 countsByYear W20780892312023 @default.
• W2078089231 crossrefType "journal-article" @default.
• W2078089231 hasAuthorship W2078089231A5017542878 @default.
• W2078089231 hasAuthorship W2078089231A5037955785 @default.
• W2078089231 hasAuthorship W2078089231A5081356989 @default.
• W2078089231 hasBestOaLocation W20780892311 @default.
• W2078089231 hasConcept C132149769 @default.
• W2078089231 hasConcept C134018914 @default.
• W2078089231 hasConcept C181199279 @default.
• W2078089231 hasConcept C184235292 @default.
• W2078089231 hasConcept C185592680 @default.
• W2078089231 hasConcept C2779395532 @default.
• W2078089231 hasConcept C2992686903 @default.
• W2078089231 hasConcept C55493867 @default.
• W2078089231 hasConcept C57074206 @default.
• W2078089231 hasConcept C58141971 @default.
• W2078089231 hasConcept C62112901 @default.
• W2078089231 hasConcept C62478195 @default.
• W2078089231 hasConcept C62826618 @default.
• W2078089231 hasConcept C86803240 @default.
• W2078089231 hasConcept C95444343 @default.

• next